Maria Costanzo writes,
> We are thinking about constructing a random shear library and have been
>experimenting with shearing total genomic DNA. Young et al. (PNAS 82:2583,
>1985) sheared DNA (0.35 mg/ml) to an average of 5 kb with 150 passages
>through a 25-gauge needle. We started with DNA at about 0.15 mg/ml and
>after 350 passagess through a 25-gauge needle, it was largely unaffected,
>with the smallest fragments about 15 kb. Does anyone have any suggestions
>about how to shear DNA efficiently ? Does the concentration affect
>shearing efficiency ? Any suggestions would be appreciated. You may
>contact me directly at mcc9 at cornell.edu, or post here; I will post a
>summary of the replies. Thank you!
As I recall from my dim dark graduate student days, the following will
improve shearing:
1. Make sure the ionic strength of your buffer is low. The lower the
salt, the more extended the DNA is.
2. Grind down your 25 guage needle so that its end is flat. When you
shear the DNA, place the end flat against a flat surface (the bottom of a
glass vial works well).
Even so, the average size of such mechanically sheared DNA is about 10 kb.
To get a smaller size, you might try a Virtis mixer or sonicating.
BTW, I have found that DNA from a roughly treated glass-bead DNA prep is
about 10 kb in lenghth. Anyone else out there have similar experiences?
Michael Lichten
lichten at helix.nih.gov
